Method of immobilizing water-soluble bioorganic compounds on a capillary-porous carrier

ABSTRACT

The method for immobilizing water-soluble bioorganic compounds to capillary-porous carrier comprises application of solutions of water-soluble bioorganic compounds onto a capillary-porous carrier, setting the carrier temperature equal to or below the dew point of the ambient air, keeping the carrier till appearance of water condensate and complete swelling of the carrier, whereupon the carrier surface is coated with a layer of water-immiscible nonluminescent inert oil and is allowed to stand till completion of the chemical reaction of bonding the bioorganic compounds with the carrier.

FIELD OF THE INVENTION

The present invention relates in general to molecular biology and morespecifically to a method for immobilizing water-soluble bioorganiccompounds onto a capillary-porous carrier.

BACKGROUND OF THE INVENTION

Known in the present state of the art are quite a number of chemicalmethods for immobilizing water-soluble bioorganic compounds, e.g.,proteins, peptides, and DNA fragments (oligonucleotides andpolynucleotides) on capillary-porous carriers. The methods are based onestablishing covalent bonds between the bioorganic compound and thecarrier. Used as the carrier are: cellulose, carboxymethylcellulose,agarose, dextran, polyaminopolystyrene, polyacrylamides and theirderivatives, and others.

One state-of-the-art method for immobilizing oligonucleotides on acapillary porous carrier, namely on a gel matrix (SU, A No. 1,794,088),consists in that the drops of a solution of oligonucleotides are appliedto an air-dried gel matrix with the aid of a micromanipulator providedwith a dispenser, whereupon the matrix with the solution ofoligonucleotides applied thereto is placed in a wet chamber for 4 hourstill completion of the reaction of bonding oligonucleotides to gel. Thenthe matrix is dried for 0.5 hour in the open air, washed with ahybridization buffer (1M NaCl, 10 mM Na₃ PO₄.7H₂ O, pH 7.0, 1 mMethylenediaminetetraacetic acid), rinsed with water, and stored dry atminus 20° C. Oligonucleotides are immobilized on a gel matrix, said gelbeing applied to the substrate, as areas (square cells) spaced from oneanother.

The chemical reaction of oligonucleotide-to-gel bonding is conducted asfollows. Used as a linking agent is 3-methyluridine bound by a 5'-3'internucleotide phosphodiester bond to the oligonucleotide to beimmobilized. 3-methyluridine is given preference due to its inability offorming strong hydrogen bonds with any naturally occurring bases.

Prior to applying the gel to the matrix, oligonucleotides containing3-methyluridine at their 3'-end are oxidized with 1 mM sodium periodatefor one hour at room temperature, precipitated with 10 volumes of 2%LiClO₄ (lithium perchlorate) in acetone, and dissolved in water.

Oxidation of oligodeoxynucleotide results in formation of a derivativecarrying a dialdehyde group at the 3'-end. Before use, the gel matrix istreated with 50% hydrazine whereby a part of the amide groups aresubstituted by the hydrazide ones which readily react with 3'-dialdehydeto yield a stable morpholine derivative.

The course of immobilization is monitored against the marker (5'-³² P)introduced with the aid of kinase, into the oligonucleotides beingimmobilized. The immobilization yield (i.e., the percentage of theoligonucleotide irreversibly bonded with the gel) is close to 80%.

The aforediscussed method, however, has a restriction on its applicationwhenever it is necessary to immobilize a great number (above 10) ofvarious nucleotides, contained in microvolumes (up to tens ofnanoliters) of solutions located in the cells (measuring up to 100 μm)of a dense polyacrylamide gel micromatrix, with the cell spacing up to200 μm when strictly single-type nucleotides are to be placed in eachcell. In this case standard conditions for the reaction of covalentbonding of oligonucleotides with the carrier are difficult to attain inall the cells of the matrix, due to inescapable partial evaporation ofsolutions in some of the cells long before the reaction is completed andoften already in the process of applying the solutions and duringtransfer of the micromatrix to a wet chamber. Thus, the quality ofimmobilization and, accordingly, that of the micromatrix are affected,the consumption of expensive reagents is increased and the processbecomes more costly.

Furthermore, the moisture-exchange process on the matrix surface becomesdifficult to control after the matrix has been placed in the wet chamberwhere water condensate may fall abundantly from the vapor-gaseous phaseupon the micromatrix surface and connect the adjoining matrix cells, orthe solution may vaporize to such an extent that the reaction wouldstop. Both such cases will result in a complete loss of the micromatrix.All discussed above shows that practical realization of the aforesaidmethod is rather complicated.

DISCLOSURE OF THE INVENTION

The present invention has for its principal object to modify theprocedures of the method for immobilizing water-soluble bioorganiccompounds on a capillary-porous carrier in such a way as to rule out apossibility of liquid evaporation during immobilization and to ensurethat covalent bonding between oligonucleotides and the gel matrixproceeds to completion, thereby rendering the process lesslabor-consuming and the technology suitable for automation and massproduction.

The foregoing object is accomplished due to the fact that in a methodproposed herein and aimed at mobilization of water-soluble bioorganiccompounds to a capillary-porous carrier, comprising application ofwater-soluble bioorganic compounds to a capillary-porous carrier, for atime necessary to complete the chemical bonding of said bioorganiccompounds with the carrier, the carrier temperature is set to be equalto or below the dew point of the ambient air, and the carrier is allowedto stand until water condensate appears and the carrier gets swollencompletely, whereupon the carrier surface is coated with a layer ofwater-immiscible nonluminescent inert oil and is let to stand until thereaction of bonding the bioorganic compounds with the carrier iscompleted.

It is preferable to use oligonucleotides or polynucleotides as thewater-soluble bioorganic compounds and make use of polyacrylamide gel asthe capillary-porous carrier.

It is also practical to keep the carrier under an oil layer for at least48 hours.

All described above makes it possible to rule out a possibility ofliquid evaporation during immobilization and to carry out the chemicalbinding of bioorganic compounds to the carrier till completion.

The method proposed herein is characterized by simplified techniquescompared with the known method, it also gives a better quality ofimmobilization and improved reproducibility.

The method proposed herein is less labor-consuming, it can be readilyautomated and may be used for mass production.

A BRIEF DESCRIPTION OF THE DRAWINGS

In the following, the invention is illustrated by a detailed descriptionof some specific embodiments thereof with reference to the accompanyingdrawings, wherein:

FIG. 1A, B, C, D is a sectional side view of a fragment of the carrier(i.e., micromatrix) shown during the process of immobilization, where:

FIG. 1A. a fragment of the micromatrix at the instant when microvolumesof the solutions of bioorganic compounds are loaded thereto;

FIG. 1B. a fragment of the micromatrix at the instant of completion ofloading;

FIG. 1C. a fragment of the micromatrix at the instant of setting itstemperature equal to or below the dew point of the ambient air;

FIG. 1D. a fragment of the micromatrix after its having been coated withthe film of an inert nonluminescent oil;

FIG. 2 presents washing curves of perfect and imperfect duplexesobtained on the micromatrix after hybridization with immobilizednucleotides.

BEST MODE OF CARRYING OUT THE INVENTION

The method proposed herein can be implemented as follows.

The microvolumes of bioorganic solutions, such as oligonucleotides,polynucleotides, water-soluble proteins, peptides, etc. are applied tothe surface of a capillary-porous carrier, that is micromatrix. Used asthe carrier can be cellulose, carboxymethylcellulose, agarose, dextran,polyaminopolystyrene, and others, though used preferably ispolyacrylamide gel.

Once the microvolumes of the solutions of bioorganic compounds have beenapplied to all cells of the matrix, the micromatrix temperature is setequal to or below the dew point of the ambient air, and is maintained sotill swelling of the gel is complete, and noncoalescent droplets ofwater condensate appear in the spacings between the cells, whereupon athin layer of an inert nonluminescent oil is cautiously applied to themicromatrix surface, the thickness of the latter layer being over 100μm. As an oil can be used purified Vaseline; phenyl (10%) methylsiliconeoil; phenyl (20%) methylsilicone oil, and others. Thereupon themicromatrix is kept under the oil layer till total completion of theimmobilization process, preferably at least for 48 hours. Then the oilis removed with a solvent, e.g. chloroform, and the matrix is dried andstored ready-for-use.

For the sake of better clarity, the process is illustrated in FIG. 1A,B, C, D, wherein a fragment of the micromatrix is shown in a sectionalside-elevation view:

FIG. 1A. At the instant when microvolumes of bioorganic solutions arebeing loaded to the micromatrix cells, the temperature of themicromatrix is maintained equal to that of the ambient air.

FIG. 1B. At the instant of completion of the loading, all droplets haveevaporated, the condition of the gel is the same in all cells.

FIG. 1C. At the instant when the water condensation from the ambient airhas been completed, the temperature of the micromatrix is below or equalto the dew point of the ambient air. The gel cells have swollen and arecoated with water condensate. Minute droplets of condensate haveappeared in the intercell spacings, said droplets not coalescing withone another.

FIG. 1D. The micromatrix is coated with the film of a nonluminescentoil, over 100 μm-thick. The temperature of the micromatrix is equal tothat of the ambient air.

The herein-proposed method is applicable for immobilizing anywater-soluble bioorganic substances to the carrier, especially in caseswhich require the presence and retention of the liquid (aqueous) phasefor a complete progress of chemical reaction of covalent bonding in thesystem `substance-carrier`.

For better understanding of the present invention a specific exemplaryembodiment thereof is given below.

EXAMPLE 1

Prepare solutions of oligonucleotides Sp-33 (3'CCGTCCAA5'), Sm-33(3'CCGTCTAA5') having a concentration of 60 pmole per μl, and containing3-methyluridine at the 3'-end, oxidate them with 1 mM sodium periodatefor one hour at room temperature and precipitate with 10 volumes of 2%LiCIO₄ in acetone, and a solution of a succinimide derivative oftetramethylrhodamine in dimethylsulfoxide with a concentration of 0.5 to1 μg per 100 μl, which serves for quality control of microdoseapplication. Using a 8% polyacrylamide gel, 30 μm-thick, a micromatrixis formed from the gel, consisting of 100×100-μm cells spaced by 200×200μm void areas, and is treated with 50% hydrazine for an hour at roomtemperature, after which microvolumes of the prepared solutions areapplied to the surface of the gel cells, namely 1.4±0.29 nl per cell(FIG. 1A). The temperature of the micromatrix is not controlled andequals the temperature of the ambient air (room temperature, FIG. 1B).After applying the microvolumes of the solutions of oligonucleotides toall cells, the temperature of the micromatrix is set to be equal to orbelow the dew point of the ambient air during the process, and ismaintained so until the gel swells completely and noncoalescent watercondensate appears in the intervals between the-cells (FIG. 1C). Thenthe surface of the micromatrix is carefully coated with a thin (over 100μm) layer of nonluminescent phenyl (10%) methylsilicone oil saturatedwith distilled water, after which the micromatrix is kept under a layerof oil at room temperature until a total completion of theimmobilization process, that is, for 48 hours (FIG. 1D). Then the oil isremoved with a solvent (chloroform), and the matrix is dried and putinto storage.

Since immobilization of bioorganic compounds on carriers is carried outwith a principal purpose of further use of thus-obtained system forspecific chemical bonding of other bioorganic compounds for theiraccretion or identification, the quality of immobilization can beassessed by an indirect method. In this particular case, such anassessment is carried out against hybridization of the preparedoligonucleotide micromatrix with a fragment of DNA fluorescent-labeledat the 5'-end and consisting of 19 bases (5'-CCTGGGCAGGTTGGTATCA-3'),which is contained in a buffer solution (1 M NaCl; 10 mM sodiumphosphate; 1 mM EDTA; pH=6.8). Then the solution (1 μl per four cells)is applied to the surface of the oligonucleotide micromatrix cooled downto 0° C. To provide a complete hybridization, the matrix is allowed tostand under these conditions for two hours, whereupon the hybridizationsolution containing DNA fragments is washed out with a cooled 1 M NaClsolution. The surface of the matrix is coated with a thin layer ofcooled 1 M NaCl solution; a cover slide is placed on the matrix and thelatter is put on a thermostated table of a fluorescence microscopeprovided with a CCD camera, and a computer-aided image processingsystem. Then a measurement cycle is conducted, the image of themicromatrix is recorded in the temperature range from 0°to 25° C. by 5°C. steps (including the margins of the range) and with a four minuteexposure at each step. Then the image is analyzed, and the signalmeasured by the CCD camera is averaged in the region corresponding toeach cell of the micromatrix, a maximum error being within 5%. Thebackground is assessed by the signals from the points situated in theimmediate vicinity of the analyzed cell, the error being within 15%. Thebackground value is subtracted from the thus-obtained signal values, andthe result is divided by the frame accumulation time (preset during themeasuring cycle). All results are expressed in percent against thebrightest cell and are graphically represented in FIG. 2B as washoutcurves of perfect (Sp-33 3'CCGTCCAA5') and imperfect (Sm-333'CCGTCTAA5') duplexes obtained on the micromatrix (four cells) afterhybridization with immobilized oligonucleotides. FIG. 2 presents theremaining duplexes as ordinate (%), and the washout temperature asabscissa (°C.).

It is known that the amount of hybridized oligonucleotide is variableand will depend (all other factors being equal) on the oligonucleotidecomposition and, when the amount of the immobilized oligonucleotide andthe composition thereof are equal, on the local conditions in a specificcell (i.e., the state of the carrier structure, accuracy of geometricsize, presence of admixtures, etc.). That is why some difference isobserved in the level of signals at the initial point (0° C.) in FIG. 2even for oligonucleotides of the same composition. Thus, the indirectmethod for assessing the immobilization quality (from hybridization)results in a slight under estimation.

It is evident from the resultant curves of Sp-33 (1, 2) and Sm-33 (1, 2)in the chart of FIG. 2 that the obtained data on hybridization and henceimmobilization are sufficient, both quantitatively and qualitatively,for unambiguously identifying the DNA fragment under test (high level ofthe initial hybridization signal at 0° C. and disappearance of thesignal for Sm-33 (1, 2) at 15° C. with a high signal level retained forSp-33 (1, 2).

Similar curves have been obtained for a great number of various duplexes(not shown), while the immobilization quality in all cases was no worsethan in the Example cited before, and the reproducibility was 100% inall experiments. This demonstrates high reproducibility and quality ofthe immobilization method being proposed herein.

Industrial Applicability

The present invention finds application in medicine, molecular biology,agriculture for genetic diagnosis, sequencing and mapping of DNA,detecting of mutations, and so on.

We claim:
 1. A method for immobilizing water-soluble bioorganiccompounds on to a carrier bondable with the compounds, comprisingapplying water-soluble bioorganic compounds onto a bondable carrierhaving a matrix of cells and kept thereon until completion of thechemical bonding between said bioorganic compounds and said carriermatrix, application of water-soluble bioorganic solutions being followedby setting the temperature of the carrier equal to or below the dewpoint of the ambient air and the carrier is allowed to stand until watercondensate swells said matrix cells, whereupon the carrier surface iscoated with a layer of water-immiscible nonluminescent inert oil andallowed to stand until the reaction of bonding the bioorganic compoundswith the carrier is completed.
 2. A method according to claim 1 whereinthe bioorganic compounds are selected from the group consisting ofoligonucleotides and polynucleotides.
 3. A method according to claim 1wherein said carrier comprises polyacrylamide gel.
 4. A method accordingto claim 1 wherein said carrier is kept under said oil layer for about48 hours.